For years, many laboratories, including ours, have focused on elucidating the molecular and cellular basis for opiate tolerance and withdrawal. Regardless of the hypotheses, one constant is that chronic drug effects are initiated by receptor signaling. Since the ?-opioid receptor (OPRM1) is a member of the rhodopsin family of G protein-coupled receptors (GPCR), the model for GPCR desensitization involving the G protein-receptor kinase (GRK) and ?-arrestin was applied to the ?-opioid receptor (OPRM1) to account for the chronic drug effect. However, other signaling pathways, such as N-methyl-D-aspartate (NMDA) receptor, protein kinase C (PKC) and even ?-opioid receptor (OPRD1), have been implicated in morphine tolerance development. The involvement of multiple protein kinases in chronic morphine effect is best exemplified by our recent observations that both Src kinase and Raf-1 kinase participate in adenyly cyclase (AC) superactivation after chronic drug treatment. These observations and others have led us to propose the hypothesis that recruitment of protein kinases by the agonist-OPRM1 complex will determine the pathway selected for cellular adaptational processes, such as opiate tolerance and withdrawal. Thus, the goals of our proposed studies are to determine whether there is an agonist-selective mechanism (i.e., protein kinase-dependent) in opiate tolerance development and whether PKC is involved in the blunting of in vivo morphine and not other agonist actions. In addition, the roles of the Src/Raf-1 kinase signaling cascade and the phosphorylation of OPRM1 in AC superactivation will be elucidated. The significance of our in vitro observations will be validated with the proposed in vivo studies. By using the approach of viral delivery of the wild type or mutant OPRM1, wild type or phosphorylation minus mutant of the PTX-insensitive Gi/o ?-subunits, or siRNA constructs to regulate the protein kinases involved, into vlPAG area of OPRM1-/- mice or double knockout mice of OPRM1 and ?Arrestin2, we will address our hypothesis with 2 specific aims: (1) To demonstrate that PKC mediates the in vivo tolerance development to morphine; (2) To delineate the pathway involved in Src-mediated OPRM1-directed AC superactivation and linking AC superactivation to naloxone precipitated withdrawal signs. We anticipate that we will demonstrate that the agonist-selective pathway in opiate tolerance development, i.e., both ?Arr and PKC pathways, are involved in tolerance development and that, by controlling the Src kinase activity within the OPRM1 signaling complex, the phosphorylation of AC by Raf-1 activated by Src leads to some if not all of the withdrawal signs observed during naloxone-precipitated withdrawal in mice chronically treated with an opioid agonist. Our proposed studies will link the action of the various protein kinases recruited and activated by OPRM1 in the behavioral responses to the chronic drug treatment. PUBLIC HEALTH RELEVANCE: Even after decades of intensive research by many laboratories, including our own, the exact detailed molecular mechanism for morphine tolerance and withdrawal remains elusive - possibly because the many mechanisms and pathways involved are different among the various opioid agonists. Our proposed studies will investigate the detailed mechanism by which the protein kinases participate in chronic drug action. From our studies, we anticipate a better understanding of molecular mechanism for morphine tolerance and dependence, eventually leading to treatment paradigms.